Engineering of Fatty Acid Pathway and Hydrocarbon Production in E. Coli

To improve the efficiency of producing fatty acid derived hydrocarbons, such as alkane, alkene, and alkyl ester, in model microorganism Escherichia coli, we are engineering pathways related to fatty acid biosynthesis using several strategies. First, a metabolic network bi-level optimization model was developed and applied to strain design for maximum fatty acid production. The gene manipulations suggested by this model were performed and led to an increase of total fatty acid from 58.5 mg/g dry cell weight in the wild type to 84.9 mg/g dry cell weight in a multiple knockout strain. Second, the triacylglycerol biosynthesis pathway was successfully introduced into E. coli by heterogeneous expression of wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase (WS/DGAT) enzymes together with the knockout of dgkA, which not only increases the total amount of fatty acids in E. coli, but also improves the fuel quality of fatty acids. Third, an alkane biosynthesis pathway of cyanobacterial origin is being explored by utilization of synthetic protein scaffolds which spatially organize the enzymes involved. All combined, these strategies should lead to the improvement of titer and yield of producing fatty acid derived hydrocarbons in microorganisms.